Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor alpha-DNA transcription complexes: analysis by microelectrospray mass spectrometry

The vitamin D receptor (VDR) binds zinc, and the activity of vitamin D depe ndent genes in cells is influenced by intracellular zinc concentrations. To determine whether zinc influences vitamin D action in cells by modulating the formation of VDR and retinoid x receptor alpha (RXR alpha) heterodimer- DNA complexes, we used microelectrospray ionization mass spectrometry (mu E SI-MS) to assess receptor-DNA interactions in the presence of varying amoun ts of zinc. In the absence of DNA, VDR and RXR alpha proteins were primaril y monomeric with small amounts of protein homodimers also observed. Zn2+ (u p to 300 muM) did not change VDR or RXR alpha monomer/homodimer ratios. Mas s spectra of VDR combined with RXR alpha were a sum of individual protein s pectral data. Zn2+ had no effect on the interactions of receptors. With inc reasing amounts of Zn2+, additional Zn2+ ions were detected bound to VDR an d RXR alpha. mu ESI-MS analyses of RXR alpha in the presence of an osteopon tin vitamin D DNA response element (OP-VDRE) showed RXR alpha homodimer/OP- VDRE complexes. DNA-protein complex formation increased on addition of Zn2 up to 200 muM; at 300 muM, Zn2+ dissociation of the RXR alpha homodimer/OP -VDRE complexes occurred, coincident with the appearance of RXRor monomeric protein. When mu ESI-MS analyses were carried out with VDR and OP-VDRE, VD R homodimer/OP-VDRE complexes were not detected. Addition of Zn2+ did not r esult in VDR/OP-VDRE complex formation. Heterodimeric VDR/RXR alpha complex es with OP-VDRE were detected by mu ESI-MS. Addition of 300 muM Zn2+ result ed in dissociation of the heterodimeric VDR/RXR alpha /OP-VDRE complex. Add ition of Mg2+ in place of Zn2+ did not alter protein/OP-VDRE complexes. Our results show that zinc modulates steroid hormone receptor-DNA interactions . Copyright (C) 2001 John Wiley & Sons, Ltd.